The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

Abstract

In recent years, there has been a noticeable increase in research interests on the Fusarium species, which includes prevalent plant pathogens and human pathogens, common microbial food contaminants and industrial microbes. Taken the advantage of gibberellin synthesis, Fusarium fujikuroi succeed in being a prevalent plant pathogen. At the meanwhile, F. fujikuroi was utilized for industrial production of gibberellins, a group of extensively applied phytohormone. F. fujikuroi has been known for its outstanding performance in gibberellin production for almost 100 years. Research activities relate to this species has lasted for a very long period. The slow development in biological investigation of F. fujikuroi is largely due to the lack of efficient research technologies and molecular tools. During the past decade, technologies to analyze the molecular basis of host-pathogen interactions and metabolic regulations have been developed rapidly, especially on the aspects of genetic manipulation. At the meanwhile, the industrial fermentation technologies kept sustained development. In this article, we reviewed the currently available research tools/methods for F. fujikuroi research, focusing on the topics about genetic engineering and gibberellin production.

Keywords: CRISPR-cas; Fusarium fujikuroi; fermentation; genetic engineering; gibberellic acid; tools.

FIGURE 1

Schematic diagram of a F. fujikuroi-rice plant infection assay.

FIGURE 2

The construct of Tet-on promoter for conditional expression in F. fujikuroi. The promoter region is composed of a tetracycline-dependent transactivator rtTA2^S-M2 (on the left of the construct, encodes rtTA protein) and an rtTA protein driven operator tetO7. The tetracycline activated rtTA protein is capable to bind the tetO7 operator and induce the targeted gene expression.

FIGURE 3

Diagram of Cas9 complex and DNA repair in genome engineering. The CRISPR-Cas system employs a short guide RNA to direct the Cas9 protein, an endonuclease, to a specific cutting site in the genome and generates DNA double strands break (DBS). The lethal DBS can be repaired by either NHEJ or HR. In the NHEJ process, proteins such as Ku70, Ku80 and Lig4 are involved. The NHEJ process results in error-prone repairs. In the HR process, usually a donor DNA is employed for precise genetic engineering.

FIGURE 4

Gibberellin biosynthesis pathway in F. fujikuroi. The involved enzymes are highlighted by boxes; arrows indicate sequential biosynthesis steps (double arrows indicate multiple reactions); the rest names are either the main intermediates or end products. The bioactive GAs are marked in bold red letters.